Puri cation and characterization of an exo-L-1,3-glucanase produced
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
FEMS Microbiology Letters 219 (2003) 81^85
www.fems-microbiology.org
Puri¢cation and characterization of an exo-L-1,3-glucanase produced
by Trichoderma asperellum
Maria Teresa F. Bara a , Adilson L. Lima b , Cirano J. Ulhoa b;
a
Faculdade de Farma¤cia, Universidade Federal de Goia¤s, 74.605-220 Goia“nia, GO, Brazil
b
Departamento de Cie“ncias Fisiolo¤gicas (ICB), Universidade Federal de Goia¤s, 74.001-940 Goia“nia, GO, Brazil
Received 29 October 2002; received in revised form 3 December 2002; accepted 6 December 2002
First published online 31 December 2002
Abstract
Trichoderma asperellum produces at least two extracellular L-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A
L-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with
9.5% yield. The molecular mass of the purified exo-L-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS^electrophoresis
slab gel. The enzyme was only active toward glucans containing L-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form
glucose. The Km and Vmax values for exo-L-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml31 and 0.246 U min31 ,
respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55‡C. Hg2þ strongly inhibited the
purified enzyme.
= 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Trichoderma asperellum ; L-1,3-Glucanase ; Regulation; Puri¢cation
1. Introduction L-1,3-Glucan is a cell-wall component of most of fungi,
and L-1,3-glucanases have been found to be directly in-
Species of the genus Trichoderma have been investigated volved in the mycoparasitism interaction between Tricho-
as biological control agents for many years. The most derma species and its host [6]. Several types of L-glucan-
common species studied were Trichoderma virens, Tricho- degrading enzymes exist, classi¢ed according to the type of
derma harzianum and Trichoderma viride [1]. T. viride was L-glucosidic linkage they cleave and mechanism of sub-
characterized by its globose, subglobose, or ellipsoidal strate attack [7]. They can hydrolyze the substrate by
warted conidia. However, two di¡erent types of warts on two possible mechanisms, identi¢ed by the products of
conidia identi¢ed as T. viride, were found and termed hydrolysis: (a) exo-L-glucanases hydrolyze the substrate
types I and II [2]. The application of combined molecular by sequentially cleaving glucose residues from the non-
data, morphology, physiology, and colony characteristics reduncing end, and (b) endo-L-glucanase cleave L-linkages
distinguished type I, which corresponds to true T. viride at random sites along the polysaccharide chain, releasing
and type II represents a new species, Trichoderma asper- smaller oligosaccharides.
ellum [2,3]. Recently some studies described the potential There is nothing in the literature about production of L-
of this species as a biological control agent against plant 1,3-glucanases by T. asperellum. However, a considerable
pathogens [4,5]. amount of research has been made with the L-1,3-gluca-
nase system of di¡erent species of Trichoderma, mainly T.
harzianum [8^12]. They have a L-1,3-glucanase secretion
system, controlled by catabolite repression and each iso-
late exhibited a di¡erent enzyme pro¢le when grown on
di¡erent carbon sources.
In this study, we report the puri¢cation and character-
* Corresponding author.
ization of an 83.1-kDa extracellular exo-L-1,3-glucanase
E-mail addresses : mbara@farmacia.ufg.br (M.T.F. Bara), produced by of T. asperellum, isolated from savanna soil
ulhoa@icb1.ufg.br (C.J. Ulhoa). of Brazil.
0378-1097 / 02 / $22.00 = 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(02)01191-6
FEMSLE 10810 6-2-0382 M.T.F. Bara et al. / FEMS Microbiology Letters 219 (2003) 81^85
2. Materials and methods tein was silver stained as described by Blum et al. [17].
Molecular mass markers were as follow: L-galactosidase
2.1. Organism and culture conditions (116 kDa), phosphorylase b (97.4 kDa), bovine serum al-
bumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase
Spores from T. asperellum, isolated from the savanna (29 kDa).
soil of the central region of Brazil (Enzymology Group Enzymatic activities in the gel were carried out as de-
collection, UFG/ICB), were collected in sterile saline, cen- scribed by Pan et al. [18]. After non-denaturing PAGE,
trifuged at 2000 rpm, washed twice and used as inoculum gels were washed with distilled water, incubated with 50
(1.0U107 spores ml31 in liquid medium^TLE). TLE me- mmol l31 sodium acetate (pH 5.0) for 60 min, and then
dium contained : 0.1% bactopeptone, 0.03% urea, 0.2% incubated with at 40‡C for 180 min in a solution contain-
KH2 PO4 , 1.4% (NH4 )2 SO4 , 0.03% MgSO4 W7H2 O, 0.03% ing 0.5% of laminarin (in 50 mmol l31 sodium acetate, pH
glucose, 0.5% Rhizoctonia solani cell wall and trace ele- 5.0). Bands with L-1,3-glucanase activity were located after
ments solution containing Fe2þ , Zn2þ , Mn2þ , Cu2þ . The boiling the gel with 2,3,5-triphenyltetrazolium chloride so-
cultures were grown in conical £asks with constant shak- lution.
ing (140 rpm) at 28‡C for 24 h. The mycelium was har-
vested by ¢ltration through ¢lter paper, and the culture 2.5. Enzyme characterization
¢ltrate was dialysed overnight against distilled water,
freeze-dried and used as source of L-1,3-glucanases. Puri- The e¡ect of pH on the enzyme activity was determined
¢cation of cell wall from R. solani was made by the meth- by varying the pH of the reaction mixtures using 100 mM
od described by Mitchell and Taylor [13]. sodium citrate (pH 2.3^4.0), 100 mM sodium acetate (pH
4.2^5.4) and 100 mM sodium phosphate (pH 5.8^7.1). The
2.2. L-1,3-Glucanase assay e¡ect of temperature on the enzymatic activity was deter-
mined at pH 5.0, in the range 25‡C to 70‡C. The e¡ects of
Enzyme activity was measured by mixing 50 Wl of sam- metallic ions and some enzyme inhibitors on L-1,3-gluca-
ple with 100 Wl of 50 mmol l31 acetate bu¡er (pH 5.0), nase activity were determined after preincubation at 50‡C
containing 0.25% laminarin (Sigma). The mixture was in- for 5 min. Michaelis^Menten constant (Km ) were deter-
cubated at 40‡C for 30 min and the reducing sugar pro- mined from Lineweaver^Burk representation of data ob-
duced was determined by the method described by Miller tained by measuring the initial rate of laminarin hydrolysis
[14]. One unit (U) of L-1,3-glucanase activity was de¢ned under the assay conditions described above and using a
as the amount of enzyme that produced 1 Wmol of reduc- range of 25^175 Wg ml31 .
ing sugar min31 under the above conditions. Protein con-
centration was determined by the method of Lowry [15], 2.6. Analysis of hydrolysis products
using bovine serum albumin as standard.
The puri¢ed L-1,3-glucanase was incubated with 2.5 mg
2.3. Enzyme puri¢cation ml31 laminarin in 100 mM sodium acetate bu¡er (pH 5.0)
at 40‡C. Sample were removed after 2, 6, 12 and 24 h
The concentrated samples were loaded on a Sephacryl incubation and hydrolysis was stopped by heating the
S-100 column (2.5U47 cm) previously equilibrated with 20 samples in boiling water for 10 min. The products were
mmol l31 Tris^HCl bu¡er pH 8.0, and eluted with the detected by thin-layer chromatography (TLC), as de-
same bu¡er at a £ow rate of 40 ml h31 . Fractions of 1.0 scribed by Lato et al. [19].
ml were collected and monitored for protein (A280 ) and L-
1,3-glucanase activity. Fractions containing L-1,3-gluca-
nase activity were pooled and applied directly onto a Q-
Sepharose Fast Flow column (1.5U6.5 cm) equilibrated
with 50 mmol l31 Tris^HCl bu¡er pH 8.0, and eluted at
a £ow rate of 60 ml h31 . The column was washed with the Table 1
E¡ect of di¡erent carbon sources on the production of L-1,3-glucanase
same bu¡er and eluted with a linear gradient of 0^0.5 mol
by T. asperellum
l31 NaCl. Fractions containing L-1,3-glucanase activity
Carbon source L-1,3-Glucanase (U mg31 )
were pooled, dialysed against water and stored at 320‡C.
Glucose 1% 0
Glucose 2% 0
2.4. Electrophoresis and enzymatic activities in gel
CWRS 0.5% 1.23 P 0.01
Chitin 0.5% 0.33 P 0.08
Polyacrylamide gel electrophoresis (SDS^PAGE) was Chitosan 0.5% 0.33 P 0.03
used to determine protein purity and the molecular mass Cellulose 0.5% 0.14 P 0.01
of the puri¢ed enzyme under denaturing conditions using Starch 0.5% 0.35 P 0.02
a 12% acrylamide gel, as described by Laemmli [16]. Pro- CWRS : Puri¢ed cell wall from R. solani.
FEMSLE 10810 6-2-03M.T.F. Bara et al. / FEMS Microbiology Letters 219 (2003) 81^85 83
1 2 3 1 2 3 4 5 6
G1
G2(β-1,3)
G2(β-1,6)
Fig. 2. Thin-layer chromatogram of products obtained from laminarin
Fig. 1. Detection of extracellular L-1,3-glucanase activity on non-dena-
and cell walls isolated of R. solani treated with the exo-L-1,3-glucanase.
turing PAGE, when T. asperellum was grown on cell walls of R. solani.
The standards used were glucose (G1), laminarobiose (G2, L-1,3) and
Lane 1, crude enzyme; lanes 2 and 3, peak 1 and 2 from gel ¢ltration
gentiobiose (G2, L-1,6) at concentration of 7 Wg. Lane 1, glucose; lane
on Sephacryl S-100, respectively.
2, laminarobiose; lane 3, gentiobiose; lane 4, puri¢ed enzyme+cell wall
isolated of R. solani; lane 5, crude enzyme+cell wall isolated of R. sola-
ni; lane 6, puri¢ed enzyme+laminarin. The ¢gure was printed from a
3. Results and discussion
scan of the TLC plate.
The e¡ects of di¡erent carbon sources on L-1,3-gluca-
nase synthesis by T. asperellum were tested in TLE me- produced by isolate T-Y [12] and seven L-1,3-glucanases
dium supplemented with glucose or one of the compounds by isolate IMI206040 [20], have been described.
listed in Table 1. Cultures were grown for 24 h, harvested We have puri¢ed a L-1,3-glucanase produced by T. as-
and analyzed for L-1,3-glucanase activity and protein. The perellum, after growth on cell wall puri¢ed from R. solani,
fungus produced L-1,3-glucanases in all carbon sources, using two steps procedures: gel ¢ltration on Sephacryl S-
but the level varied depending on the carbon source 100 and ion exchange chromatography on Q-Sepharose.
used (Table 1). The highest activity was obtained when Gel ¢ltration resulted in the separation of two peaks of
cell walls puri¢ed from R. solani were used as substrate proteins with L-glucanase activity, and the ¢rst peak was
(1.23 U mg31 ), and no activity was detected in glucose- used for further puri¢cation of the enzyme on a Q-Seph-
containing medium. Signi¢cant levels of L-1,3-glucanase arose. The enzyme was puri¢ed 35-fold with a recovery of
activity were also found in presence of chitin (0.33 U 9.5% (Table 2).
mg31 ), chitosan (0.33 U mg31 ) and starch (0.35 U SDS^PAGE showed that the enzyme migrated as a sin-
mg31 ), but the levels found were almost the same. High gle band with an estimated molecular mass of 83.1 kDa
levels of L-1,3-glucanase activity in the culture-containing (Table 4). The molecular masses of L-1,3-glucanases pro-
cell walls puri¢ed from R. solani, suggest that the regula- duced by Trichoderma appear to vary considerably, not
tion of L-1,3-glucanase expression in T. asperellum was only between organisms, but also within the same species.
also in£uenced by the levels of L-glucan present in the Molecular masses of exo-L-1,3-glucanases from T. harzia-
inducer as described by isolates of T. harzianum [9,10,20]. num T-Y were in a similar range of 75 [12], while a smaller
To determine which secreted protein corresponded to L- 29-, 31- and 40-kDa exo-L-1,3-glucanase also have been
1,3-glucanases, we assayed for enzyme activity by per- isolated and characterized [8,21,22].
forming non-denaturing PAGE (Fig. 1). In the presence The puri¢ed enzyme appeared to act in an exo-gluca-
of cell walls puri¢ed from R. solani, at least two bands nase-like fashion, as indicated by the release of glucose
with L-1,3-glucanase activity were detected and separated from laminarin and puri¢ed cell walls from R. solani, after
by gel ¢ltration on Sephacryl S-100 (Fig. 1). The produc- 24 h incubation (Fig. 2). The enzyme also released gentio-
tion of three L-1,3-glucanases by T. harzianum isolate TC biose from laminarin, suggesting that the commercial sub-
[9], four L-1,3-glucanases by isolate CECT2413 [10], ¢ve strate contains a L-1,6-glucosidic linkage (Fig. 2, lane 6).
Table 2
Summary of the puri¢cation steps of the exo-L-1,3-glucanase produced by T. asperellum
Step Total protein (mg) Total activity (U) Speci¢c activity (U mg31 ) Puri¢cation (fold) Yield (%)
Crude enzyme 1.497 0.325 0.217 1 100
Sephacryl S-100 0.024 0.049 2.041 9.4 15.1
Q-Sepharose 0.004 0,031 7.750 35.7 9.5
Typical values are given.
FEMSLE 10810 6-2-0384 M.T.F. Bara et al. / FEMS Microbiology Letters 219 (2003) 81^85
Exo-L-1,3-glucanases are enzymes that hydrolyze the L- Table 4
Biochemical properties of the puri¢ed exo-L-1,3-glucanase from T. as-
glucan chain by sequentially cleaving glucose residues
perellum
from the non-reducing end, and have been described in
many fungi [7], including some produced by T. harzianum Molecular mass (kDa) 83.1 P 2.3
pH optimum 5.1 P 0.2
[8,21,22,23]. Temperature optimum (‡C) 55.0 P 2.0
To establish the speci¢city of exo-L-1,3-glucanases Temperature stability
against a variety of glucan substrates, laminarin, puri¢ed pH 5.0/45‡C/90 min 85 P 2.5%
cell walls from R. solani, chitin, chitosan, cellulose and pH 5.0/50‡C/90 min 57 P 1.2%
starch were used as substrates (Table 3). The puri¢ed en- Km (mg ml31 ) 0.087
Vmax (U min31 ) 0.246
zyme was active only toward glucans containing L-1,3 Inhibition by Hg2þ 100%
linkages, such as laminarin, and puri¢ed cell walls from
R. solani (Table 3). The enzyme hydrolyzed laminarin
more readily than puri¢ed cell wall, but did not attack ity. However, further study will be required to determine if
cellulose, chitin, chitosan or starch. Inactivity of the en- other enzymes are involved in the hydrolysis of cell walls
zyme towards cellulose suggested that the enzyme was from R. solani.
unable to cleave L-1,4 linkages within the L-glucan mole-
cule.
The apparent Km (0.087 mg ml31 ) of the exo-L-1,3-glu- Acknowledgements
canases from T. asperellum was substantially lower than
those reported for T. harzianum (2.1 mg ml31 ) [21], T. This work was supported by a biotechnology research
harzianum TC (1.72 mg ml31 ) [8], T. harzianum CECT grant to C.J.U (CNPq and FUNAPE/UFG). M.T.B was
2413 (3.3 mg ml31 ) [10], but was similar to that reported supported by CoordenacTa‹o de AperfeicToamento de Pes-
for T. harzianum TY (0.1 mg ml31 ) [12]. soal de N|¤vel Superior do Brasil (CAPES).
The e¡ects of pH and temperature on the enzyme activ-
ity are shown in Table 4. The optimal pH for the enzyme
activity (5.1) was similar to that found for exo- and endo-
L-1,3-glucanases from a variety of T. harzianum strains References
[8,11,21,23]. The optimal activities of fungal L-1,3-gluca-
[1] Hermosa, M.R., Grondona, I., Iturriaga, E.A., Diaz-Minguez, J.M.,
nases are usually in the range of 4.0 and 6.0 [7]. The Castro, M., Monte, E. and Garcia-Acha, I. (2000) Molecular char-
optimum temperature for the enzyme activity was found acterization and identi¢cation of biocontrol isolates of Trichoderma
to be 55‡C at pH 5.0 (Table 4), and it is in agreement with spp. Appl. Environ. Microbiol. 66, 1890^1898.
exo-L-1,3-glucanases from T. harzianum TC [8]. The en- [2] Meyer, R.J. and Plaskowitz, J.S. (1989) Scanning electron microsco-
py of conidia and conidial matrix of Trichoderma. Mycologia 81,
zyme was stable for at least 90 min when incubated at
312^317.
45‡C and retained 57% of maximum activity at 50‡C (Ta- [3] Lieckfeldt, E., Samuels, G.J., Nirenberg, H.I. and Petrini, O. (1999)
ble 4). Hg2þ strongly inhibited enzyme activity (Table 4). A morphological and molecular perspective of Trichoderma viride: is
The complete inhibition by mercuric ions may indicate the it one or two species? Appl. Environ. Microbiol. 65, 2418^2428.
importance of indole amino acids in enzyme function, as [4] Kullnig, C.M., Krupica, T., Woo, S.L., Mach, R.L., Rey, M., Beni-
tez, T., Lorito, M. and Kubicek, C.P. (2001) Confusion abounds over
has been demonstrated for an exo- and endo-L-1,3-gluca-
identities of Trichoderma biocontrol isolates. Mycol. Res. 105, 769^
nase from T. harzianum TC [8,23]. 772.
In conclusion, T. asperellum produces at least two pro- [5] Cotxarrera, L., Trillas-Gay, M.I., Steinberg, C. and Alabouvette, C.
teins with L-1,3-glucanase activity when induced with cell (2001) Use of sewage sludge compost and Trichoderma asperellum
wall isolated from R. solani. An 83.1-kDa exo-L-1,3-glu- isolates to suppress Fusarium wilt of tomato. Soil Biol. Biochem.
34, 467^476.
canase was puri¢ed and exhibited high a⁄nity for the
[6] Harman, G.E. (2000) Myths and dogmas of biocontrol: changes in
substrate laminarin, and it had a reasonable thermostabil- perceptions derived from research on Trichoderma harzianum T-22.
Plant Dis. 84, 377^393.
[7] Pitson, S.M., Seviour, R.J. and McDougall, B.M. (1993) Noncellu-
Table 3 lolytic fungal L-glucanases: their physiology and regulation. Enz.
Substrate speci¢city of exo-L-1,3-glucanases from T. asperellum Microbiol. Technol. 15, 178^192.
Substrate (0.5%) Main linkage/monomer Relative activity (%) [8] Noronha, E.F. and Ulhoa, C.J. (2000) Characterization of a 29-kDa
L-1,3-glucanase from Trichoderma harzianum. FEMS Microbiol. Lett.
Laminarin L-1,3-/glucose 100
183, 119^123.
CWRS L-1,4- L-1,3-/GlcNAc; glucose 25
[9] Noronha, E.F., Kipnis, A., Junqueira-Kipnis, A.P. and Ulhoa, C.J.
Cellulose L-1,4-/glucose 0
(2000) Regulation of a 36-kDa L-1,3-glucanase synthesis in Tricho-
Chitin L-1,4-/GlcNAc 0
derma harzianum. FEMS Microbiol. Lett. 188, 19^22.
Chitosan L-1,4-/GlcN 0
[10] De La Cruz, J., Pintor-Toro, J.A., Benitez, T., Llobell, A. and Ro-
Starch L-1,4-; L-1,6/glucose 0
mero, L. (1995) A novel endo-L-1,3-glucanase, BGN13.1, involved in
Results are means values of three replicates. CWRS: Puri¢ed cell wall the mycoparasitism of Trichoderma harzianum. J. Bacteriol. 177,
from R. solani. GlcNAc : N-acetylglucosamine. GlcN: Glucosamine. 6937^6945.
FEMSLE 10810 6-2-03M.T.F. Bara et al. / FEMS Microbiology Letters 219 (2003) 81^85 85
[11] El-Katatny, M.H., Gudelj, M., Robra, K.-H., Elnaghy, M.A. and [18] Pan, S.Q., Ye, X.S. and Kue, J. (1989) Direct detection of L-1,3-
Gubitz, G.M. (2001) Chacterization of a chitinase and an endo- L- glucanase isozymes on polyacrylamide electrophoresis and isoelectro-
1,3-glucanase from Trichoderma harzianum Rifai T24 involved in focusing gels. Anal. Biochem. 182, 136^140.
control of the phytopathogen Sclerotium rolfsii. Appl. Microbiol. [19] Lato, M., Brunelli, B. and Ciu⁄ni, G. (1969) Thin layer chromatog-
Biotechnol. 56, 137^143. raphy of carbohydrates on silica gel impregnated with sodium ace-
[12] Ramot, O., Cohen-Kupiec, R. and Chet, I. (2000) Regulation of L- tate, monosodium phosphate and disodium phosphate. J. Chroma-
1,3-glucanase by carbon starvation in the mycoparasite Trichoderma togr. 39, 407^417.
harzianum. Mycol. Res. 104, 415^420. [20] Vazquez-Garciduenas, S., Leal-Morales, C.A. and Herrera-Estrella,
[13] Mitchell, A.D. and Taylor, I.E. (1969) Cell-wall proteins of Aspergil- A. (1998) Analysis of the L-1,3-glucanolytic system of the biocontrol
lus niger and Chaetomium globosum. J. Gen. Microbiol. 59, 103^109. agent Trichoderma harzianum. Appl. Environ. Microbiol. 64, 1442^
[14] Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for the deter- 1446.
mination of reducing sugar. Anal. Chem. 31, 426^428. [21] Kitamoto, Y., Kono, R., Shimotori, A., Mori, N. and Ichikawa, Y.
[15] Lowry, O.H., Rosebrough, N., Farr, A. and Randall, R. (1951) Pro- (1987) Puri¢cation and some properties of an exo-L-1,3-glucanase
tein measurement with the Folin Phenol Reagent. J. Biol. Chem. 193, from Trichoderma harzianum. Agric. Biol. Chem. 51, 3385^3386.
265^275. [22] Dubourdieu, D., Desplanques, C., Villettaz, J. and Ribereau-Gayon,
[16] Laemmli, U.K. (1970) Cleavage of structural proteins during assem- P. (1985) Investigations of an industrial L-D-glucanase from Tricho-
bly of the head of the bacteriophage T4 . Nature 227, 680^685. derma harzianum. Carbohydr. Res. 144, 277^287.
[17] Blum, H., Beier, H. and Gross, H. (1987) Improved silver staining of [23] Noronha, E.F. and Ulhoa, C.J. (1996) Puri¢cation and characteriza-
plant proteins, RNA and DNA in polyacrylamide gels. Electropho- tion of an endo-L-1,3-glucanase from Trichoderma harzianum. Can. J.
resis 8, 93^99. Microbiol. 42, 1039^1044.
FEMSLE 10810 6-2-03You can also read
